Caspase Dependent and Independent Anti-hematological Malignancy Activity of AMHA1 Attenuated Newcastle Disease Virus

Hematological malignancies remain one of the leading causes of death worldwide despite advances in cancer therapeutics. Newcastle disease virus (NDV) is a member of Paramyxoviridae that elicits considerable interest as an anticancer agent because it can replicate up to 10 000 times faster in human cancer cells than in most normal cancer cells. Several NDV strains reportedly induce the cytolysis of cancerous cell lines. The attenuated Iraqi strain (AMHA1) of NDV is a novel oncolytic agent with promising antitumor characteristics, including apoptosis induction. This study aimed to evaluate the ability of the AMHA1 NDV strain to induce apoptotic cell death in hematological tumors through caspase-dependent or independent apoptotic pathways. The cytolytic effects of AMHA1 NDV strains of different multiplicity of infection (MOIs) (20, 15,10, 5, 3, 1, 0.5, and 0.1 )and exposure for all hematological malignancy cell lines (human non-Hodgkin lymphoma SR and human multiple myeloma (COLO 677) and human monocytic leukemia THP1) have been determined through a microtetrazolium (MTT) assay. Propidium iodide and acridine orange (AO/PI) double staining were used to examine the ability of attenuated NDV strain to induce apoptosis in infected cells under a fluorescence microscope and to quantify the percentage of apoptosis induction. Quantitative immunocytochemistry assay was further used to study the caspase-dependent and independent protein expression levels in infected and control cells. Cells treated with NDV strains showed a higher cell-death percentage than untreated cells as quantified by the MTT assay. AO/PI results revealed that NDV exerted a powerful and significant effect on apoptosis induction (P<0.0001) in the human cancer cell lines tested in comparison with control cells. Immunocytochemistry in AMHA1 NDV- infected human hematological cell lines revealed a remarkable increase in the expression of caspase 8, 9 (dependent pathway), apoptosis-inducing factor, and endonuclease G (independent pathway) in comparison with untreated cells. This study demonstrated the role of the Iraqi NDV strain in inducing apoptosis through dependent and independent pathways in cancer cells and thus its high potential as an antitumor agent.

ematological malignancies are a collection of heterogeneous conditions that originate in bone marrow cells and the lymphatic system. Three groups, namely, leukemia, lymphoma, and plasmacell neoplasms, are available, and their overall incidence is rising (1), Malignancies are still the leading cause of death despite advances in surgery, chemotherapy, and radiotherapy that is characterized by low efficacy and high toxicity for the patient (2). Virotherapy has become a promising option, as the viral replication provides continuous amplification of the injected dose, which continues until stopped by the immune response, and it enters and destroys cancer cells without affecting surrounding normal cells (3,4). Some viruses, such as Newcastle disease virus (NDV), exhibit the ability to destroy cancer cells. These viruses can be used without any genetic manipulation for increasing selectivity in animal models and human clinical trials (5). NDV can replicate up to 10 000 times better in human cancer cells than in most normal cells (6). NDV causes fatal disease in birds; however, in humans, it is generally not very virulent, causing only mild flu-like symptoms, conjunctivitis, and laryngitis (7). The Avulavirus genus-attenuated AMHA1 Iraqi ND strain belongs to the Paramyxoviridae family, and the NDV genome is a single-stranded negative-sense RNA of 15 186 nucleotides (8). The avian NDV is a naturally oncotropic RNA virus (9). Many studies were conducted worldwide using different strains of NDV on different cancer cell lines in vitro and in vivo. The most important strains were strain 73-T, Lytic strain Italians, NDV strain P701, and NDV strain Ulster. Local NDV strains AF2240 and V4 UPM as oncolytic agents have been studied on breast cancer cell lines (MCF-7 and MDA-231) and brain tumor cell lines (DBTRG.5MG and U87MG) (10)(11)(12)(13). NDV replicates rapidly and infects neighboring tumor cells through the release of progeny virions (7). Furthermore, NDV kills tumor cells by modification of the tumor-cell surface and generation of specific cellular immune responses (14,15). Moreover, NDV can kill tumor cells through induction of apoptosis in the infected cells (16,17). NDV replication is independent of the host-cell DNA replication that is the target of most chemotherapeutic drugs and radiotherapy and that makes NDV a candidate oncolytic agent to break resistance of tumor cells to the therapies (5).
The present work aims to determine the involvement of a caspase-dependent or independent apoptotic pathway induced by NDV.  (21).

Cytotoxicity assay
The cells were seeded in 96-well plates and were washed with phosphate-buffered saline (PBS) before inoculating with and without the attenuated

Quantification of the apoptosis proteins
The adherent cell line (SR) wascultured on coverslips. The cells were allowed to develop a monolayer. Then, NDV (IC 50 ) was exposed for 72 h. Subsequently, the cells were fixed with cold acetone for 2-5 min, then removed using cold acetone, and washed with PBS thrice, and left to dry. After fixation, the slides were incubated in a humidified chamber with 1% H 2 O 2 for 10 min, washed two to three times with PBS, and incubated with 1.5% blocking reagent for 30-40 min at room temperature. Then, the primary antibody of the CAS-8, CAS-9, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA) was washed two to three times with PBS for 1-1:30 h.
Afterward, the secondary antibody was added and allowed to stand for 2 h. The secondary antibody was then stained by the ImmunoCruz™ mouse   In this study, a fluorometric cell viability assay by using AO/PI was carried out to detect the  DNase activity (37), AIF was thought to be working together with endonuclease G to promote DNA degradation (38).
It was also shown previously that the NDVinduced caspase-dependent and independent pathways, AIF, and Endo G could induce caspaseindependent apoptotic cell death by directly mediating chromatinolysis (39). This effect allows mitochondrial dysfunction to orchestrate cell death even in the absence of caspase activation (40).
In conclusion, our study suggests that NDV is a very promising anti-hematological malignancy agent that was found to act by inducing apoptosis through caspase-dependent and caspase-independent pathways of apoptosis.